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First published online 23 May 2007
doi: 10.1242/dev.02862
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1 Department of Developmental Neurobiology, Tohoku University, Graduate School
of Medicine, Seiryo-machi 2-1, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
2 PRESTO, Japan Science and Technology Corporation, Japan.
3 Department of Developmental Neuroscience, Center for Translational and
Advanced Animal Research on Human Diseases, Tohoku University, Graduate School
of Medicine, Seiryo-machi 2-1, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
4 Department of Molecular Biomedical Sciences, College of Veterinary Medicine,
North Carolina State University, Raleigh, NC 27606, USA.
* Author for correspondence (e-mail: wakasama{at}mail.tains.tohoku.ac.jp)
Accepted 12 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Asymmetric cell division, Numb, Transitin, Nestin, Neuroepithelium, Chick
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For example, basal daughter cells that receive Prospero and
Numb differentiate into ganglion mother cells, whereas the apical daughter
cell remains as a neuroblast. It is essential, therefore, that intracellular
localization of the fate determinants and the plane of cell cleavage be
coordinated.
In vertebrate nervous system development, previous studies have suggested
that the plane of cell cleavage in mitosis could be correlated with the
behavior of the daughter cells in NE (Chenn
and McConnell, 1995; Cayouette
et al., 2001). For example, in neuroepithelial cells (NE cells) of
the new-born ferret neocortex region, when the cell cleavage plane is parallel
to the ventricular surface, while the apical daughter cell appears to remain
undifferentiated in the ventricular zone, the basal daughter cell rapidly
migrates basally and may undergo neuronal differentiation
(Chenn and McConnell, 1995). By
contrast, cell division with the cleavage plane perpendicular to the
ventricular surface results in a symmetric NE cell phenotype
(Chenn and McConnell, 1995).
Such observations, along with the asymmetric localization and segregation of
fate determinants in Drosophila neurogenesis, implied that the
mechanisms involved in the fate determination of daughter cells of neural stem
cells in vertebrates might be similar to those in Drosophila.
Consistent with this inference, in vertebrates, Numb protein localizes
asymmetrically in mitotic NE cells (Zhong
et al., 1996; Wakamatsu et
al., 1999; Cayouette et al.,
2001; Shen et al.,
2002), as well as in mitotic cells of dorsal root ganglia (DRG)
(Wakamatsu et al., 2000).
Thus, depending on the orientation of cell cleavage, Numb can be inherited
unevenly by daughter cells. It has been shown that Numb binds to the
intracellular domain of Notch and antagonizes Notch activation
(Guo et al., 1996;
Wakamatsu et al., 1999). Thus,
one of the functions of Numb is to regulate the differentiation of neural
cells by modulating Notch signaling (for a review, see
Cayouette and Raff, 2001).
Consistently, conditional and non-conditional knockouts of Numb and
numb-like in mice reveal severe defects in the maintenance of NE and in
neurogenesis (Zhong et al.,
2000; Zilian et al.,
2001; Peterson et al., 2002; Peterson et al., 2004;
Li et al., 2003).
In the developing central nervous system of avian embryos, Numb protein
localizes in the basal cortex of the mitotic NE cells
(Wakamatsu et al., 1999). As
Numb is a cytoplasmic adapter protein, it must itself be anchored to cellular
architectural components to localize asymmetrically in mitotic cells. In this
study, we show that transitin, a type IV intermediate filament protein,
colocalizes with Numb in the cortex of mitotic NE cells. Our biochemical
assays and RNA interference (RNAi) analyses indicate that transitin associates
directly with Numb, and provides an anchor site enabling Numb to localize in
the cell cortex. These observations prompted us to follow the localization and
segregation of transitin-EGFP in live NE cells, and we found that basally
localized transitin moved laterally during mitosis. Thus, the lateral shift of
transitin enables its asymmetric segregation to one of the daughter cells even
when the cleavage plane is perpendicular to the ventricular surface.
Consistent with the important role(s) of Numb in neurogenesis, transitin
knockdown by RNAi promoted neuronal differentiation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wakamatsu et al., 1997;
Wakamatsu et al., 2000).
Polyclonal anti-chick Numb (Wakamatsu et
al., 1999; Wakamatsu et al.,
2000); monoclonal 7B3 (Henion
et al., 2000) and EAP-300 anti-transitin
(McCabe et al., 1992;
Cole and Lee, 1997); and 16A11
anti-Hu (Marusich et al.,
1994; Wakamatsu and Weston,
1997) antibodies were described previously. Anti-FLAG (M2,
Invitrogen), anti-vimentin (VIM3B4, Roche), anti-
-tubulin (GTU-88,
Sigma), anti-phospho-vimentin (polyclonal, SantaCruz), anti-GFP (polyclonal,
Chemicon), anti-neuron-specific type III ß-tubulin (TuJ1, BAbCO) and
anti-BrdU (BMC9318, Roche) were obtained commercially. TUNEL staining for
detection of cell death was performed as described previously
(Wakamatsu et al., 1998). For
photographing and cell counts, individual cell was carefully examined on a
standard fluorescence microscope (Axioplan 2, Zeiss) that was equipped with a
cooled CCD camera (AxioCam, Zeiss) by changing the focal plane to ensure the
examined cells were intact.
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). For co-immunoprecipitation assay, GST-Numb(158-582) and Mal-Transitin(328-816) [Mal-Trans(328-816)] were purified with glutathione- and maltose-affinity columns, respectively. Purified proteins were mixed in a binding buffer [50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 mM NaCl, 10% glycerol, 0.5 mg/ml BSA, 5 mM ß-mercaptoethanol] at 4°C for 1 hour. Anti-GST rabbit antibody was added and the mixture was further incubated at 4°C for 1 hour. Finally, protein A resin was added to the mixture and the suspension was incubated at 4°C for 1 hour. After extensive washing in phosphate-buffered saline (PBS), the resin was collected by a brief centrifugation, and the resin was suspended in PBS. Bound proteins were eluted in SDS-PAGE sample buffer at 90°C for 10 minutes. Elutes were subjected to standard SDS-PAGE.
Electroporation into chick embryonic spinal cord
Expression vectors of N-terminally FLAG-tagged transitin were constructed
in pyDF30 (Wakamatsu and Weston,
1997). pEGFP-N1, pEGFP-F and pd1EGFP were obtained from
BD-Clontech. pRCAS(B) was a gift from Koji Tamura (Tohoku University, Sendai,
Japan). For sustained, low levels of Trans(1-327)-d1EGFP expression,
Trans(1-327) was connected in frame to d1EGFP (unstable
mutant of EGFP with the attached PEST sequence), and subcloned into
pRCAS(B) for a relatively weak promoter activity of the long terminal repeat
(LTR), and genome integration. Electroporation into the neural tube of chick
embryos was performed basically as described
(Funahashi et al., 1999;
Suzuki et al., 2006). For a
knockdown of transitin, corresponding RNA (sense:
5'-CCCAUUGCAAUGAGCCAGGTT-3', antisense:
5'-CCUGGCUCAUUGCAAUGGGTT-3') was generated and annealed (Custom
siRNA Synthesis, Takara), and injected into the lumen of the neural tube,
along with the EGFP expression vector, for electroporation. For a
short hairpin (sh)RNA-mediated knockdown of transitin, DNA sequence,
corresponding to transitin cDNA 289-309
(5'-CCAGGGACAACCTGTATGAGG-3'), was inserted to pSilencer (Ambion)
to generate pSilencer-Trans289. For negative control experiments, a
mutated sequence
(5'-CCAGGTACCACCTGGATAAGG-3';
mutations are underlined) was used for construction of
pSilencer-Trans289m. BrdU pulse-labeling was performed as previously
described (Wakamatsu et al.,
2000).
Time-lapse imaging of cultured chick brain slices
Embryonic day (E)3 chick forebrain was electroporated with expression
vectors of RCAS(B)-Trans(1-327)-d1EGFP and pDsRed2-nuc (purchased
from Clontech, carrying a red fluorescent protein gene, DsRed2, fused
to a nuclear localization signal of Large T antigen), E5 transfected
telencephalon were sliced to 200 µm thickness with vibratome (MICROSLICER
DTK-3000W, D.S.K.), embedded in a collagen gel, and Dulbecco's modified
Eagle's medium (DMEM)/F12 medium containing 10% fetal calf serum and 5% horse
serum was overlaid. Slices were observed in a CO2 incubating
chamber at 38°C set on an inverted fluorescence microscope (Axiovert,
Zeiss), with a cooled CCD camera (AxioCam). Normal rate of NE cell
proliferation and differentiation, as well as normal neuronal layer formation,
were confirmed by BrdU uptake and immunostaining with anti-Hu antibody after
24 hours of culture (data not shown).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), showed
an overlapping intracellular localization with Numb in interphase processes
and in the basal cortex of mitotic NE cells in various regions of the
developing central nervous system (Fig.
1A,B), as well as in the cortex of mitotic cells in the developing
DRG (Fig. 1C). Because 7B3
antibody has been shown to recognize transitin, an intermediate filament
protein (Henion et al., 2000),
we tested another monoclonal antibody (EAP-300), which was independently
raised against transitin (McCabe at al.,
1992; Cole and Lee,
1997). This antibody gave identical immunoreactivity in mitotic NE
cells (data not shown), thereby confirming the asymmetrical localization of
transitin. Detailed observation of Numb and transitin staining revealed a
faithful colocalization throughout the M phase of NE cells
(Fig. 2A). For asymmetrically
localizing factors to be segregated unevenly to daughter cells after cell
division, the localization of such factors and the cleavage orientation will
have to be coordinated. In this paper, we classified the orientation of the
cleavage plane (and metaphase plate) into three groups, according to Sanada
and Tsai (Sanada and Tsai,
2005). Thus, cells with vertical cleavage have a mitotic spindle
parallel to the ventricular surface (60-90°), cells with horizontal
cleavage have spindle that are perpendicular to the surface (0-30°), and
intermediate cells have spindle in between (30-60°). In the case of
mammalian neocortical region (Chenn and
McConnell, 1995; Sanada and
Tsai, 2005), a significant proportion of mitotic NE cells showed a
horizontal and intermediate cleavage plane (e.g. approximately 20% and 30%,
respectively, in E15 mouse cortex) (Sadana and Tsai, 2005). We thus examined
the orientation of the cleavage plane of E4 and E5 telencephalon (pallium
region), and of E4 metencephalon (ventral half) of chick embryos because
active neurogenesis appeared to occur at these stages, based on the expression
of neuronal markers. Transverse sections were double-stained with DAPI
(chromosomes) and anti--tubulin antibody for spindle poles (data not
shown, but see Fig. 2B). With a
significant contrast to the proportion observed in mammals
(Chenn and McConnell, 1995;
Sanada and Tsai, 2005),
approximately 14% and 5% of mitotic NE cells showed an intermediate and a
horizontal cleavage plane, respectively, and more than 80% of mitotic NE cells
were classified as vertical. Although a slightly angled cleavage plane could
be sufficient to unevenly segregate fate determinants localized in the narrow
apical endfoot, if any are localized there (see
Kosodo et al., 2004), basal
localization of Numb and transitin in the cell cortex seemed to be too wide.
However, both Numb and transitin were often asymmetrically segregated to one
of the daughter cells in telophase (Fig.
2A). Thus, the localization of Numb and transitin was further
examined in relation to the cleavage plane and to the stages of M phase in
mitotic NE cells of E4 pallium and ventral metencephalon
(Fig. 2B-D). In both cases, the
proportion of NE cells with asymmetrically-localized Numb and transitin
increased in later M phase (approximately 60% and 35% of telophase NE cells in
the telencephalon and metencephalon, respectively, see also
Fig. 2C,D), suggesting basal
Numb and transitin were relocated laterally during the M phase. Interestingly,
the proportion of asymmetrically localized Numb and transitin is higher in the
telencephalon than that in the metencephalon
(Fig. 2C,D), which might have
reflected upon the rate of neurogenesis at the stages examined.
|
).
Because of the large size of transitin
(Yuan et al., 1997), pieces of
transitin cDNA were subcloned into bacterial expression vectors
(Fig. 3A) to obtain GST-fusion
of the transitin recombinants. Expression of GST-transitin fusions was
confirmed by Coomassie Blue staining (Fig.
3B) and by western blotting with anti-GST antibody (data not
shown). Membrane-transferred fusion proteins were overlaid with lysates
prepared from Numb-transfected NIH3T3 cells. Binding of Numb to a
fragment of a linker sequence between the rod and the heptad repeat domains of
transitin (Yuan et al., 1997),
Trans(328-816) (Fig. 3B), was
detected by anti-Numb antibody. Because this experiment did not show whether
the binding of Trans(328-816) and Numb was direct or indirect, we performed a
similar assay with a recombinant MBP-fusion of Trans(328-816) and recombinant
GST-fusion of Numb, and a series of Numb deletion mutant proteins
(Fig. 3C). Anti-GST antibody
detected the binding of Numb to Trans(328-816)
(Fig. 3D), suggesting that the
interaction of transitin and Numb was direct. Furthermore, deletions in the
Numb construct (Fig.
3C) revealed the interaction to be via the carboxy sequence of
Numb and not the phospho-tyrosine binding (PTB) domain
(Fig. 3D). The direct binding
of Trans(328-816) and Numb was further confirmed by co-immunoprecipitation
assay (Fig. 3E, see also
Materials and methods). Thus, affinity-purified Trans(328-816)-MBP and
GST-fusion of the C-terminal half of Numb [Numb(158-582)] were pre-incubated
in solution, and GST-Numb(158-582) was immunoprecipitated with anti-GST
antibody. Co-precipitated Trans(328-816)-MBP could be detected with anti-MBP
antibody.
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The requirement of transitin for the basal localization of Numb was further confirmed by knockdown of transitin with RNAi (Fig. 4E,F). Double-stranded (ds)RNA, corresponding to the transitin mRNA sequence (see also Materials and methods), was electroporated into the dorsal neural tube of E2 chicken embryos, along with an expression vector of EGFP to visualize transfected cells. At 16 hours after transfection, compared with neural tube cells transfected with EGFP alone or with EGFP and mutated dsRNA, endogenous transitin immunoreactivity was clearly reduced in the neural tube cells co-transfected with EGFP and transitin double-strand RNA (Fig. 4E). Under this condition, localization of Numb in the basal and basolateral cortex of EGFP-transfected prophase and metaphase NE cells was examined (Fig. 4F). Transfection of EGFP alone or its co-transfection with mutated RNA showed no difference in the localization of Numb. Thus, 94.7% (n=95) of EGFP-transfected cells and 93.1% (n=72) of cells co-transfected with mutated dsRNA revealed clear basal or basolateral cortical localization of Numb. By contrast, transitin gene knockdown resulted in a severe reduction of basal or basolateral Numb immunoreactivity in the cell cortex (Fig, 4E), and only 31.6% of transfected mitotic NE cells (n=80) showed such localization of Numb. These observations suggested that, in mitotic NE cells of chick embryos, transitin localized in the basal cortex and provided anchor sites for Numb.
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). In fact, full-length transitin required either
vimentin or desmin to participate in stable intermediate filament formation,
unless its C-terminal tail was removed. Because vimentin is expressed in NE
and is colocalized in the radial processes of interphase NE cells
(Fig. 5A), we compared the
intracellular localization of vimentin and transitin in mitotic NE cells.
vimentin colocalized basally with transitin from prophase to metaphase
(Fig. 5A,B). However, from
anaphase, basal localization of vimentin was no longer observed and, instead,
punctate vimentin immunoreactivity was observed in the cytoplasm
(Fig. 5B). This observation
contrasted with the persistent cortical localization of transitin throughout
mitosis (Fig. 5C,D, lower
panels). It has been shown previously that intermediate filament proteins,
such as vimentin, are phosphorylated in M phase, and such phosphorylation
leads to the disassembly of the intermediate filaments
(Ando et al., 1989;
Goto et al., 1998).
Consistently, phosphorylated vimentin was observed in M-phase NE cells
(Fig. 5C,D upper panels).
Interestingly, phospho-vimentin (Ser39) was seen in the basal cortex and in
the cytoplasm (Fig. 5C),
whereas phospho-vimentin (Ser72) was detected throughout the cytoplasm from
prophase to metaphase (Fig.
5D). Phosphorylation of vimentin and dissociation from the basal
cortex suggested that, in the basal cortex of mitotic NE cells, transitin no
longer participates in the rigid intermediate filament structure and that
transitin-Numb complexes may be present in a more flexible form in the cell
cortex.
Dynamic movement of transitin in mitosis
As described above, endogenous Numb-transitin complexes appeared to be
relocated during mitosis. Therefore, we were motivated to examine the
orientation of cell cleavage and the movement of Numb-transitin during mitosis
of NE cells in live embryos. As indicated above, FLAG-tagged Trans(1-327)
could localize in the basal cortex of mitotic NE cells when the expression
level was relatively low (Fig.
4D), probably by participating in intermediate filament structure.
We generated an expression vector of d1EGFP-fusion of
Trans(1-327) (see Materials and methods), and found that
Trans(1-327)-d1EGFP fusion proteins faithfully colocalized in the basal cortex
of mitotic NE cells with endogenous transitin
(Fig. 6A) and Numb
(Fig. 6B). We then transfected
this construct into the dorsal telencephalon (pallium region) of E3 chick
embryos, and slices of the transfected telencephalon were prepared 2 days
later. The localization of Trans(1-327)-d1EGFP was then examined in slice
culture (see Materials and methods). An expression vector of DsRed2
with a nuclear localization signal (pDsRed2-nuc, see Materials and methods)
was sometimes co-transfected, allowing chromosomes on the metaphase plate and
the cleavage orientation to be observed, when co-expressed. Out of 53 mitotic
NE cells examined, only one of them revealed a nearly horizontal cleavage
plane (Table 1). Consistent
with the observation in fixed tissues (see above), in 52 mitotic cells, the
cleavage plane was almost vertical (Table
1). In 18 cases of the 52 mitotic cells vertically dividing,
Trans(1-327)-d1EGFP was not actively transported, and was segregated evenly to
both of the daughter cells (Table
1). In the remaining 34 cases, the fusion protein appeared to
shift laterally from late metaphase to early anaphase
(Fig. 6C, and see Movie 1 in
the supplementary material), during which time Trans(1-327)-d1EGFP would be
allowed to segregate to one of the daughter cells
(Fig. 6C). However, unlike
endogenous transitin, the asymmetric cortical localization of
Trans(1-327)-d1EGFP was gradually lost during anaphase, suggesting a
disassembly of intermediate filament (see above). Interestingly, we
occasionally observed that oblique metaphase plate orientation rotates along
with Trans(1-327)-d1EGFP during mitosis of NE cells
(Fig. 6C), as observed in the
mouse cortex (Sanada and Ysai,
2005).
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) and Hu
RNA-binding protein 16A11 (Marusich et
al., 1994; Wakamatsu and
Weston, 1997) was also examined both 24 and 48 hours after
transfection (Fig. 7F,G).
Although no significant change in neurogenesis ahead of schedule was observed
24 hours after transfection of Trans289, most transfected cells
expressed Hu (Fig. 7F) and TuJ1
(data not shown), and intermingled with untransfected neurons in the neuronal
layer (Fig. 7F). Thus,
transitin knockdown inhibited proliferation and promoted neuronal
differentiation. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Sanada and Tsai,
2005), although it remains to be seen whether such angled cell
cleavage will indeed result in the uneven segregation of fate determinants. In
avian brains, more than 80% of NE cells show a vertical cleavage plane,
similar to that in the neural retina
(Silva et al., 2002). Although
the cleavage plane and the fate of daughter cells has not yet been correlated
in avian systems, because Numb and transitin can be segregated asymmetrically
even with vertical cleavage, the rate of asymmetric cell division can be
underestimated in experiments if only the cleavage plane is used as an
indicator of asymmetric cell division.
Transitin anchors Numb to the basal cortex
Previously, we have reported that Numb localizes in the basal cortex of
mitotic NE cells (Wakamatsu et al.,
1999). In this study, we show that the intermediate filament
protein transitin provides an anchor site for Numb in the basal cortex of
mitotic NE cells. How transitin initially localizes to the basal cortex of
prophase NE cells is not clear, but it was previously reported that transitin
mRNA is preferentially transported to the basal processes of interphase NE
cells (Lee and Cole, 2000). It
is therefore possible that locally translated transitin in the basal processes
may be transported to the cortex prior to mitosis. Consistent with this idea,
in our time-lapse analysis we often observed that Trans(1-327)-d1EGFP in the
basal process moved apically prior to the M phase (Y.W. and N.N., unpublished
observation).
The regulatory mechanisms of asymmetric cell division have been extensively
studied in Drosophila nervous system development, and Numb localizes
asymmetrically in mitotic neural cells (for a review, see
Roegiers and Jan, 2004).
Despite many similarities in vertebrates and invertebrates in the regulatory
mechanism of development, however, chick and Drosophila now appear to
have some differences, because the genome project of Drosophila
showed that this widely used experimental animal does not have cytoplasmic
intermediate filaments (Rubin et al.,
2000). Even in vertebrates, the molecular machinery to control
Numb localization does not appear to be conserved, because mouse Numb
localizes in the apical side of NE cells
(Zhong et al., 1996), whereas
avian Numb localizes in the basal cortex (this study) (see also
Wakamatsu et al., 1999).
Nestin is the closest relative of transitin, because they are categorized in
the same intermediate filament subclass due to the sequence homology in their
rod domain, and because they are expressed in NE cells and muscle precursors
(for a review, see Herrmann and Aebi,
2000). However, the sequence of the C-terminal tail, which is
responsible for Numb-transitin association, is not conserved in nestin (Y.W.,
unpublished data), and, more importantly, nestin is not asymmetrically
localized in mitotic NE cells (YW, unpublished observation). Although it is
not known whether nestin is involved in neurogenesis, for reasons mentioned
above, nestin does not seem to directly regulate Numb localization in mouse NE
cells.
Lateral transport of transitin in mitosis
In this study, we show that, even if the vertical cleavage plane would
result in a horizontal cell division, such cells can still segregate
Numb-transitin complexes asymmetrically, because these components, anchored
within the basal cortex, shift laterally in late M phase, and thereby allow
preferential segregation into one of the two daughter cells. It remains to be
studied how the lateral transport of Numb-transitin complexes is regulated.
Because, in a third of NE divisions, Trans(1-327)-d1EGFP still remained in the
basal cortex and segregated symmetrically, some unknown mechanism(s) probably
determines whether the lateral transport during M phase is initiated. It is of
note that, at the early phase of mitosis in NE cells, vimentin is
phosphorylated, which probably leads to the dissociation of the intermediate
filament structure. It has been shown that such phosphorylation-dependent
dissociation of intermediate filaments in the M phase is important for
cytokinesis (Ando et al., 1989;
Goto et al., 1998) and that
Aurora B activity, which is strictly regulated during M phase, is involved in
this process (Goto et al.,
2003). Thus, dissociation of rigid intermediate filament structure
of vimentin-transitin by phosphorylation of vimentin in the early M phase may
permit the transport of transitin. Such dissociation of intermediate filament
in M phase is consistent with the fact that cortically-localized
Trans(1-327)-d1EGFP becomes cytoplasmic in the late M phase.
Transitin in neurogenesis
As mentioned above, Numb has been shown to regulate neurogenesis
in mouse embryos, both positively and negatively
(Zhong et al., 2000;
Zilian et al., 2001; Peterson
et al., 2002; Peterson et al., 2004; Li et
al., 2003). The cause of such discrepancy is unclear, but changes
in the expression of Numb isoforms during development
(Bani-Yaghoub et al., 2007)
might explain the differences observed between mouse knockout lines, at least
in part. In any case, the requirement of transitin for the proper
intracellular localization of Numb suggests the involvement of transitin in
the neurogenesis of avian embryos. Consistently, transitin knockdown causes a
depletion of NE cells by reducing proliferation and promoting neuronal
differentiation, although how the reduction of transitin expression causes
such a phenotype remains elusive. One possibility is that, because transitin
stabilizes Numb (see above), transitin knockdown may lead to the reduction of
Numb protein, which would otherwise inhibit neurogenesis. This idea is
consistent with the observation that Numb-knockout mice show
precocious neurogenesis (Zhong et al.,
2000; Peterson et al., 2002; Peterson et al., 2004).
Alternatively, by losing the transitin anchor, a release of functional Numb in
the cytoplasm may promote neurogenesis, possibly by inhibiting Notch
signaling. This idea is consistent with the decreased neurogenesis observed in
certain Numb-knockout mouse lines
(Zilian et al., 2001;
Li et al., 2003). Because a
recent study (Zhou et al.,
2007) suggests that Numb may also influence neurogenesis
independently of Notch signaling, it seems important to knockdown
Numb in avian system in order to compare the phenotype with that of
mouse knockouts. Nevertheless, transitin is unique, because no other
intermediate filament protein has been shown to regulate neurogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2425/DC1
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ACKNOWLEDGMENTS |
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